Staphylococcus aureus causes a range of life-threatening diseases and is a leading cause of infections in the United States. The arrival of antibiotic-resistant strains, such as deadly methicillin-resistant S. aureus (MRSA) has made it crucial to understand the molecular mechanisms of pathogenesis. Iron is an essential nutrient for S. aureus growth and is actively procured from humans during an infection. Heme-iron within Hemoglobin (Hb) is preferentially used by S. aureus as an iron source. It is acquired by S. aureus using nine Iron regulated surface determinant (Isd) proteins that first capture Hb on the cell surface and extract its heme. The heme is then passed across the cell wall via a series of hemoreceptors, imported into the cytoplasm, and degraded to release free iron. A Ruth L. Kirschstein National Research Service Award to Promote Diversity in Health- Related Research will fund research to study how the Isd-system in S. aureus captures and extracts heme from Hb. The funding will also provide extensive training in a wide range of advanced modern structural, biochemical, and cellular methods. Research will focus on the staphylococcal Hb receptor IsdH. In aim #1, the structure of the receptor in its apo-state will be determined using a combination of newly developed paramagnetic relaxation enhancement (PRE) and residual dipolar coupling (RDC) NMR methods, enabling a model of the heme extraction process from Hb to be formulated. In aim #2, the mechanism of heme extraction from Hb will be probed using targeted amino acid mutagenesis as well as in vitro and in vivo heme transfer experiments. Finally, in aim #3, the molecular basis through which IsdH transfers captured heme to downstream hemoreceptors within the Isd-system that are embedded in the cell wall will be investigated. The transient, ultra-low affinity interaction of heme transfer can only be studied using NMR chemical perturbation and PRE experiments. The Isd-system is required for S. aureus virulence and related systems are present in a number of other clinically important pathogens. The results of this research could therefore facilitate the development of anti-infective agents that work by inhibiting heme acquisition.